This proposal focuses on efficiently targeting critical protein-protein interactions required for WASF3 stability using stapled peptides (SPs) as a means of suppressing breast cancer cell invasion and metastasis as a proof- of-principle and may also apply broadly to other cancers that depend on WASF3 for invasion and metastasis. Women with metastatic cancers have limited treatment options and shorter lifespans compared to those with indolent tumors. Current understanding suggests that successful metastatic colonization in distant organs depends on expression of the WASF3 gene. A critical barrier to progress in improving treatment options for metastasis is the lack of conventional drugs that target the metastasis process. The goal of this project is to develop innovative and optimized targeting strategies to block WASF3 function and so inhibit invasion and metastasis in breast cancer cells. An emerging approach to suppress protein function is by targeting protein interactions essential for the function of the target using stapled peptides (SPs). SPs contain modified amino acids that maintain a stable alpha helix conformation that is resistant to proteolysis, promotes active cellular uptake and is not immunogenic. WASF3 is normally held in an autorepressed conformation through interactions with CYFIP1 and NCKAP1, which are essential components of the WASF Regulatory Complex (WRC). Knockdown of any of these proteins leads to destabilization of the WASF3 complex and suppression of invasion and metastasis. Preliminary data shows that targeting the CYFIP1-WASF3 and CYFIP-NCKAP1 interactions using SPs leads to suppression of invasion in vitro and metastasis in vivo. These prototype SPs could be used in preclinical studies but it has been shown that a wide range of modification of SPs can significantly improve their efficacy. Our objectives include optimizing these SPs to increase their solubility, specificity and stability using design and medicinal chemistry approaches to develop more potent biologics and determine the most effective derivatives that suppress metastasis. The optimized SPs will then be used in in vivo models of metastasis using a variety of mouse models of metastatic breast cancer to evaluate their ability to suppress metastasis. We will also use a zebrafish model of intravasation to test the hypothesis that WASF3, through its control of MMPs and NFkB signaling, is vital for invasion into blood vessels. We have already shown that minor modifications of active peptides increase their effectiveness in vitro. These proof-of- principle experiments demonstrate that SPs can target intracellular proteins and may serve as an effective strategy to dissect and inhibit tumor metastasis. The impact of these studies is based on the knowledge of the fundamental biology behind WASF3 function allowing the use of second-generation peptide technology to target the highly specific functions of WASF3. As a result, the development of improved therapeutic approaches for the treatment of the metastatic disease will have profound implications for the clinical management of breast (and potentially other) cancer patients.